Wet Lab

Read excellent literatures about synthetic biology and projects on iGEM Official Website.

Wet Lab

We discussed with our instructors about the project designing and decided to improve Saccharomyces cerevisiae surface display technology. Then we read iGEM projects about Saccharomyces cerevisiae surface display.

Wet Lab

We had a brainstorm about yeast surface display.

Wet Lab

After discussion, we set our initial project theme that displaying human tubulins on the surface of Saccharomyces cerevisiae. Moreover, we read more literatures about tubulins.

Wet Lab

We visited Mr Meng who is the doctor of Chinese Academy of Sciences and discussed our project with him. Through the discussion, we found it difficult to keep tubulins lasting polymerization in vitro. So we attempted to look for more stable materials. At last, we found flagellin which can polymerize spontaneously in vitro.

Wet Lab

Our experiment was discussed and designed. We divided the display system into two parts: the display modules and the secretory modules. With the special structure of surface display system, we could display the particular subunit onto the Saccharomyces cerevisiae surface as a linkage site and secrete other subunits into the extracellular environment to create a subunit-rich surroundings. With the optimal concentration of subunits, GTP and other ions, those subunits could reconstitute into microtubules and flagellum. In this way, we could finally display the microtubule and flagellum on the cell surface. And then, we could use this upgraded system to facilitate small molecules screening.

Dry Lab

Built a mathematical model to test the theoretical possibility of polymerization for tubulins and flagellins in vitro.

Wet Lab

In microtubule part, we intended to construct 8 vectors, they are pYD1-α-tubulin、pYD1-β-tubulin、pYCα-α-tubulin、pYD1-mCherry(positive control)、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-mGFP、pYCα-mCherry(positive control).

Searched DNA sequences (FliC、PETase、XynA、BG、EG、CBH)

Dry Lab

Tried to find the most appropriate length for linker to improve the possibility of polymerization.

Wet Lab

Obtained the target genes sequence(α-tubulin、β-tubulin、mCherry、GFP)and plasmids (pYD1:used to display, pYCα：used to secrete). We designed primers by using snapgene and sent them to the company for synthesis.

Designed and optimized DNA sequences of flagellin and enzymes(PETase、XynA、BG、EG、CBH). Send them to America for synthesis.

Dry Lab

We studied the mechanisms of tubulin and flagellin polymerization.

Wet Lab

Plasmids were digested by enzymes, and we had an agarose gel electrophoresis to prove true.

We used PCR to amplify target genes (α-tublin、β-tublin、mCherry、GFP) and used agarose gel electrophoresis to prove true. Then, we utilized infusion technology to ligate target genes with pYD1 or pYCα，and had an agarose gel electrophoresis to prove true. Moreover, we transformed vectors we constructing into competent Escherichia coli cells(DH5α). Screened on LB plate(added Ampicillin). Sent to the company to make sure the sequence.

Dry Lab

We discussed the main modeling problem in our project.

Wet Lab

As sequencing results show, we had successfully constructed pYD1-α and pYD1-mCherry. We continued constructing the rest plasmids ( pYD1-β-tubulin、pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-mGFP、pYCα-mCherry).

Dry Lab

We learned as many as possible modeling which maybe useful for our work.

Wet Lab

According to the growing curve we measured, Saccharomyces cerevisiae competent cells were made up, and we attempted to transform recombined plasmids in it using chemical transforming method.

Continued constructing plasmids.

Dry Lab

We learned as many as possible modeling which maybe useful for our work.

Wet Lab

We successfully constructed recombined plasmids(pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-mCherry). We constructed pYD1-β once again.

Continued transforming recombined plasmids in Saccharomyces cerevisiae (EBY100 & IVSCc1) competent cells using chemical transforming method.

Dry Lab

We discussed to make sure what problem we were about to solve.

Wet Lab

Obtained the synthetic DNA flagellins and enzymes including PETase、XynA、BG、EG and CBH.

We prepared for the Final Exam so we stopped the experiments.

Dry Lab

We discussed to make sure what method we were about to use and what model we were about to build.

Wet Lab

pYD1-β was constructed successfully.

Plasmids were digested by enzymes, and we had a gel electrophoresis to prove true.

To get recombined plasmids(pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG)、pYCα-FliC(CBH)) containing corresponding enzyme, we fused FliC-N respectively with five enzymes(PETase、XynA、BG、EG、CBH) through PCR to get a part of target gene. Then we infused the part of target gene with FliC-C and plasmids digested by enzymes. These plasmids could express secretory proteins. Screened strains on LB plate (added Ampicillin). Sent sequencing.

Recombined plasmid pYD1-FliC-Mgfp was constructed in the same way with pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG)、pYCα-FliC(CBH).

We tried electronic transformation way to transform plasmids into yeast competent cells due to the fact that we always failed in chemical transformation way.

Dry Lab

We established models using statistical mechanics describing the process of tubulin polymerization in vitro.

Wet Lab

Sent recombined plasmids including pYDF-eGFP、pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG) and pYCα-FliC(CBH) sequencing.

We succeeded in electronic transformation way, transforming pYD1-mCherry into yeast competent cells and screening on SD plate (added Leu).

Dry Lab

Built a mathematical model to show the process of polymerization for flagellins in vitro.

Wet Lab

We transformed pYD1-mCherry into yeast competent cells EBY100 in electronic transformation way and screened on SD plate (added Leu), expanding culture.

Plasmids ( pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG)、pYCα-FliC(CBH))had successful sequenced.

Reconstructed pYDF-eGFP.

Dry Lab

After collecting data, we constructed a three-dimensional model of tubulin polymerization, in which the independent variables were temperature, concentration, and energy GTP.

Wet Lab

We transformed pYD1-α-tubulin、pYD1-β-tubulin into yeast competent cells EBY100 successfully.

We constructed pYDF-eGFP successfully.

We transformed pYDF-eGFP into yeast competent cells EBY100 in electronic transformation way and screened on SD plate (added leu), expanding culture.

Wet Lab

We transformed plasmid pYD1-mCherry (positive) into Saccharomyces cerevisiae EBY100 and observed that the extracted protein have red fluorescence. The expression of target protein in EBY100 was successfully. A strong reducing agent DDT was added to the cell culture medium of EBY100 to break the disulfide bond. Then the target protein mCherry displayed on the Saccharomyces cerevisiae surface was separated from Saccharomyces cerevisiae EBY100, the protein in the supernatant was extracted and the fluorescence was observed.

Wet Lab

We transformed pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-mGFP、pYCα-mCherry (positive) into yeast competent cells INVSc1 that was cultured on SD plates including leucine, tryptophan, histidine to screen.

A strong reducing agent DDT was added to the cell culture medium of EBY100 to break the disulfide bond. Then we extracted the Saccharomyces cerevisiae EBY100 which were successfully transformed in plasmids pYDF-eGFP. The protein in the supernatant was extracted and use coomassie blue staining to prove true. The protein was successfully extracted and the coomassie blue staining experiment succeeded.

Wet Lab

We successfully transformed plasmids(pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-mCherry) into INVSc1.

Wet Lab

Saccharomyces cerevisiae that was transformed in plasmids(pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-mCherry) successfully expressed the target protein.

Wet Lab

Through coomassie blue staining, target proteins expressed by pYD1-α-tubulin、pYD1-β-tubulin were successfully tested . Western Blot was used in chemical coloration to confirm the target protein.

We constructed models to simulate the actual working state of Tubulin and Flagellum.

Wet Lab

The effect of substrate DAB staining on protein detection was not ideal in Western blot. We attempted to use a more sensitive ECL color-developing method and achieved successfully.

Saccharomyces cerevisiae that was transformed in plasmids(pYD1-α-tubulin、pYD1-β-tubulin) successfully expressed the target protein in cells.

Western Blot in ECL color-developing method confirmed the target protein was expressed.

Wet Lab

We transformed pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-mGFP、pYCα-mCherry (positive) into yeast competent cells INVSc1 that was cultured on SD plates including leucine, tryptophan, histidine to screen.

Wet Lab

Through coomassie blue staining, target proteins expressed by pYCα-α-tubuin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-mGFP、pYCα-mCherry (positive) were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.

Wet Lab

Through coomassie blue staining, target proteins expressed by pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG)、pYCα-FliC(CBH) were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.

Wet Lab

Through coomassie blue staining, target proteins expressed by pYCα-FliC(BG)、pYCα- FliC(EG)、pYCα-FliC(CBH) were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.

Dry Lab

The effect of tubulin concentration and GTP concentration on the length and intensity of microtubule polymerization was analyzed by establishing ordinary differential equations.

Wet Lab

Through coomassie blue staining, target proteins expressed by pYCα-FliC(PETase)、pYCα-FliC(XynA) were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.

Wet Lab

Make the standard curve of XynA enzyme activity.
The activity of XynA enzyme expressed by pYCα-FliC(XynA) was successfully tested.
Purified proteins for polymerization in vitro.

Dry Lab

Simulation of the efficiency of flagellin aggregation on yeast surface by flagellin concentration and yeast concentration.

Wet Lab

The activity of XynA enzyme expressed by pYD1-FliC(XynA) was successfully tested.
Western Blot in ECL color-developing method confirmed the target protein (expressed by pYCα-FliC (XynA) and pYCα-mcherry) purified from supernatant.
Attempt to polymerize microtubule and flagellar filaments in vitro.
The polymerized microtubules on yeast surface were observed with High Resolution Transmission Electron Microscopy (HRTEM).

Dry Lab

The relationship between flagellin polymerization and time was used to guide the culture time of yeast.